A Three-Year Survey on the Worldwide Occurrence of Mycotoxins in Feedstuffs and Feed

Between January 2009 and December 2011, a total of 7049 corn, soybean/soybean meal, wheat, dried distillers grains with solubles (DDGS) and finished feed samples were analyzed for the occurrence of aflatoxins (Afla), zearalenone (ZEN), deoxynivalenol (DON), fumonisins (FUM) and ochratoxin A (OTA). Samples were sourced in the Americas, Europe and Asia. Afla, ZEN, DON, FUM and OTA were present respectively in 33%, 45%, 59% 64% and 28% of analyzed samples between 2009 and 2011. From the 23,781 mycotoxin analyzes performed, 81% were positive for at least one mycotoxin. Results of this survey are provided by calendar year, in order to potentially show different trends on mycotoxin occurrence in distinct years: by commodity type and within the same commodity, and by region, to potentially reveal differences in mycotoxin contamination in commodities sourced in diverse regions.     

Mycotoxin Co-Occurrence in Michigan Harvested Maize Grain

Mycotoxins are secondary metabolites produced by fungi that, depending on the type and exposure levels, can be a threat to human and animal health. When multiple mycotoxins occur together, their risk effects on human and animal health can be additive or synergistic. Little information is known about the specific types of mycotoxins or their co-occurrence in the state of Michigan and the Great Lakes region of the United States. To understand the types, incidences, severities, and frequency of co-occurrence of mycotoxins in maize grain (Zea mays L.), samples were collected from across Michigan over two years and analyzed for 20 different mycotoxins. Every sample was contaminated with at least four and six mycotoxins in 2017 and 2018, respectively. Incidence and severity of each mycotoxin varied by year and across locations. Correlations were found between mycotoxins, particularly mycotoxins produced by Fusarium spp. Environmental differences at each location played a role in which mycotoxins were present and at what levels. Overall, data from this study demonstrated that mycotoxin co-occurrence occurs at high levels in Michigan, especially with mycotoxins produced by Fusarium spp., such as deoxynivalenol.     

Mycotoxin Exposure in Children through Breakfast Cereal Consumption in Chile

Mycotoxins are unavoidable contaminants produced by fungi in food, especially grains. This study aimed to measure the occurrence and levels of total aflatoxins (AFs); ochratoxin A (OTA); zearalenone (ZEN); fumonisins B1, B2, and B3 (FUM); deoxynivalenol (DON); and T-2/HT-2 toxins in the four most commonly consumed breakfast cereals in Chile and to assess mycotoxin exposure and risk in children aged 2 to 13 years due to cereal consumption. In this study, a total of 110 batches with three subsamples of the four brands were sampled in supermarkets from November 2019 to June 2021. Samples were analyzed by Veratox^® ELISA (Neogen). Exposure was assessed by estimated daily intake (EDI) considering the levels found in a modified lower bound (mLB) and upper bound (UB). Risk was estimated by margin of exposure (MOE) in the case of OTA and AFs and hazard quotient (HQ) for the rest of the mycotoxins. No T2/HT2 toxins were detected. Few samples had quantifiable levels of ZEN, FUM, and DON except for brand 1, with a mean (standard deviation, SD) of 54 (20), 1552 (351), and 706 (218) ng/g, respectively. In addition, three FUM samples and one DON sample had values over the Chilean regulation. Brands 2, 3, and 4 had quantifiable levels of AFs, with mean (SD) values of 1.3 (0.1), 2.1 (0.6), and 1.9 (0.4) ng/g, respectively. Brand 3 had quantifiable levels of OTA, with a mean (SD) of 2.3 (0.4) ng/g. Estimated exposure indicated a risk of AFs in all scenarios, and of FUM for brand 1 consumption, OTA and DON for brand 3 consumption, and OTA for brand 4 consumption in the mLB worst-case scenario. In general, mycotoxin levels were below the Chilean regulatory limits, but most of them were above the EU regulation for processed cereal-based food in young children. Because the risk was higher in the 2- to 5-year-old children, we recommend special regulations for this group in Chile.     

Effect of Popcorn (Zea mays var. everta) Popping Mode (Microwave,Hot Oil,and Hot Air) on Fumonisins and Deoxynivalenol Contamination Levels

Mycotoxins are secondary metabolites that are produced by molds during their development. According to fungal physiological particularities, mycotoxins can contaminate crops before harvest or during storage. Among toxins that represent a real public health issue, those produced by Fusarium genus in cereals before harvest are of great importance since they are the most frequent in European productions. Among them, deoxynivalenol (DON) and fumonisins (FUM) frequently contaminate maize. In recent years, numerous studies have investigated whether food processing techniques can be exploited to reduce the levels of these two mycotoxins, which would allow the identification and quantification of parameters affecting mycotoxin stability. The particularity of the popcorn process is that it associates heat treatment with a particular physical phenomenon (i.e., expansion). Three methods exist to implement the popcorn transformation process: hot air, hot oil, and microwaves, all of which are tested in this study. The results show that all popping modes significantly reduce FUM contents in both Mushroom and Butterfly types of popcorn. The mean initial contamination of 1351 µg/kg was reduced by 91% on average after popping. For DON, the reduction was less important despite a lower initial contamination than for FUM (560 µg/kg). Only the hot oil popping for the Mushroom type significantly reduced the contamination up to 78% compared to unpopped controls. Hot oil popping appears to provide the most important reduction for the two considered mycotoxins for both types of popcorn (−98% and −58% average reduction for FUM and DON, respectively).     

Evaluation of a Yeast Hydrolysate from a Novel Strain of Saccharomyces cerevisiae for Mycotoxin Mitigation using In Vitro and In Vivo Models

Mycotoxicoses in animals are caused by exposure to mycotoxin-contaminated feeds. Disease risk is managed using dietary adsorbing agents which reduce oral bioavailability. The objective of this work was to evaluate the efficacy of three selected yeast products as mycotoxin binders using in vitro and in vivo models. Their capacity to adsorb deoxynivalenol (DON), zearalenone (ZEA), and ochratoxin A (OTA) was evaluated using an in vitro model designed to simulate the pH conditions during gastric passage in a monogastric animal. Results showed that only one product, an enzymatic yeast hydrolysate (YHY) of a novel strain Saccharomyces cerevisiae, adsorbed about 45% of DON in solution. Next, we determined the effect of YHY on oral absorption of a DON, ZEA, and OTA mixture using a toxicokinetic model in swine. Toxicokinetic modeling of the plasma concentration-time profiles of DON, OTA, and zearalenone-glucuronide (ZEA-GlcA) showed that YHY tended to reduce the maximal plasma concentration of OTA by 17%. YHY did not reduce oral bioavailability of OTA, DON, and ZEA-GlcA. Within the context of this experiment, and despite some positive indications from both the in vitro and in vivo models employed, we conclude that the YHY prototype was not an effective agent for multiple mycotoxin adsorption.     

Modelling the Renal Excretion of the Mycotoxin Deoxynivalenol in Humans in an Everyday Situation

The dietary exposure to the mycotoxin deoxynivalenol (DON) can be assessed by human biomonitoring (HBM). Here, we assessed the relation between dietary DON intake and the excretion of its major metabolite DON-15-glucuronide (DON15GlcA) through time, in an everyday situation. For 49 volunteers from the EuroMix biomonitoring study, the intake of DON from each meal was calculated and the excretion of DON and its metabolites was analyzed for each urine void collected separately throughout a 24-h period. The relation between DON and DON15GlcA was analyzed with a statistical model to assess the residence time and the excreted fraction of ingested DON as DON15GlcA (f_{abs_excr}). F_{abs_excr} was treated as a random effect variable to address its heterogeneity in the population. The estimated time in which 97.5% of the ingested DON was excreted as DON15GlcA was 12.1 h, the elimination half-life was 4.0 h. Based on the estimated f_{abs_excr}, the mean reversed dosimetry factor (RDF) of DON15GlcA was 2.28. This RDF can be used to calculate the amount of total DON intake in an everyday situation, based on the excreted amount of DON15GlcA. We show that urine samples collected over 24 h are the optimal design to study DON exposure by HBM.     

Effects of Deoxynivalenol and Mycotoxin Adsorbent Agents on Mitogen-Activated Protein Kinase Signaling Pathways and Inflammation-Associated Gene Expression in Porcine Intestinal Epithelial Cells

Deoxynivalenol (DON) is the most prevalent mycotoxin in swine feedstuffs. The intestinal epithelial cells represent the first target for the DON. Here, we studied the effects of DON and mycotoxin adsorbent agents on mitogen-activated protein kinase (MAPK) signaling pathways and inflammation-associated gene expression in porcine intestinal epithelial cells (IPEC-J2). Results showed that phosphorylation of MAPK signaling pathways (p38, ERK, and JNK) was increased after treatment of DON or lipopolysaccharide (LPS) in IPEC-J2 cells. The phosphorylation of p38, ERK, and JNK was not further enhanced after co-treatment with DON and LPS. The inos and cox-2 mRNA expression were significantly induced at 6 h after treatment of DON. DON treatment significantly increased the claudin 3 and occludin mRNA expression at 12 h. DON in combination with LPS treatment did not further increase the inflammation and tight junction-associated gene expression. The DON-induced phosphorylation of MAPK signaling pathways was impaired by mycotoxin adsorbent agent (nanoscale silicate platelets and the mixture of montmorillonites and yeast cell walls) treatment, thereby decreasing inflammation and tight junction-associated gene expression. Taken together, these findings demonstrate that DON triggers the inflammation in IPEC-J2 cells by phosphorylation of MAPK signaling pathways and LPS does not further augment the DON-induced inflammatory responses. Mycotoxin adsorbent agents can attenuate DON-induced inflammatory responses in IPEC-J2 cells through modulation of the phosphorylation of p38, ERK, and JNK.     

Role of Sesamia nonagrioides and Ostrinia nubilalis as Vectors of Fusarium spp. and Contribution of Corn Borer-Resistant Bt Maize to Mycotoxin Reduction

Maize expressing Cry1Ab insecticidal toxin (Bt maize) is an effective method to control Sesamia nonagrioides and Ostrinia nubilalis, the most damaging corn borers of southern Europe. In this area, maize is prone to Fusarium infections, which can produce mycotoxins that pose a serious risk to human and animal health, causing significant economic losses in the agrifood industry. To investigate the influence of corn borer damage on the presence of Fusarium species and their mycotoxins, Bt maize ears and insect-damaged ears of non-Bt maize were collected from commercial fields in three Bt maize growing areas in Spain, and differences in contamination were assessed. Additionally, larvae of both borer species were collected to evaluate their role as vectors of these molds. Non-Bt maize ears showed significantly higher presence of F. verticillioides, F. proliferatum, and F. subglutinans than Bt maize ears. For the first time, Fusarium species have been isolated from larvae of the two species. The most frequently found mycotoxins in ears were fumonisins, with non-Bt ears being significantly more contaminated than those of Bt maize. High levels of fumonisins were shown to correlate with the occurrence of corn borers in the ear and the presence of F. verticillioides and F. proliferatum.     

Deoxynivalenol: Toxicology,Degradation by Bacteria,and Phylogenetic Analysis

Deoxynivalenol (DON) is a toxic secondary metabolite produced by fungi that contaminates many crops, mainly wheat, maize, and barley. It affects animal health, causing intestinal barrier impairment and immunostimulatory effect in low doses and emesis, reduction in feed conversion rate, and immunosuppression in high doses. As it is very hard to completely avoid DON’s production in the field, mitigatory methods have been developed. Biodegradation has become a promising method as new microorganisms are studied and new enzymatic routes are described. Understanding the common root of bacteria with DON degradation capability and the relationship with their place of isolation may bring insights for more effective ways to find DON-degrading microorganisms. The purpose of this review is to bring an overview of the occurrence, regulation, metabolism, and toxicology of DON as addressed in recent publications focusing on animal production, as well as to explore the enzymatic routes described for DON’s degradation by microorganisms and the phylogenetic relationship among them.     

The Toxicological Impacts of the Fusarium Mycotoxin,Deoxynivalenol, in Poultry Flocks with Special Reference to Immunotoxicity

Deoxynivalenol (DON) is a common Fusarium toxin in poultry feed. Chickens are more resistant to the adverse impacts of deoxynivalenol (DON) compared to other species. In general, the acute form of DON mycotoxicosis rarely occurs in poultry flocks under normal conditions. However, if diets contain low levels of DON (less than 5 mg DON/kg diet), lower productivity, impaired immunity and higher susceptibility to infectious diseases can occur. The molecular mechanism of action of DON has not been completely understood. A significant influence of DON in chickens is the impairment of immunological functions. It was known that low doses of DON elevated the serum IgA levels and affected both cell-mediated and humoral immunity in animals. DON is shown to suppress the antibody response to infectious bronchitis vaccine (IBV) and to Newcastle disease virus (NDV) in broilers (10 mg DON/kg feed) and laying hens (3.5 to 14 mg of DON/kg feed), respectively. Moreover, DON (10 mg DON/kg feed) decreased tumor necrosis factor alpha (TNF-α) in the plasma of broilers. DON can severely affect the immune system and, due to its negative impact on performance and productivity, can eventually result in high economic losses to poultry producers. The present review highlights the impacts of DON intoxication on cell mediated immunity, humoral immunity, gut immunity, immune organs and pro-inflammatory cytokines in chickens.     

Current Situation of Mycotoxin Contamination and Co-occurrence in Animal Feed—Focus on Europe

Mycotoxins are secondary metabolites produced by fungi especially those belonging to the genus Aspergillus, Penicillum and Fusarium. Mycotoxin contamination can occur in all agricultural commodities in the field and/or during storage, if conditions are favourable to fungal growth. Regarding animal feed, five mycotoxins (aflatoxins, deoxynivalenol, zearalenone, fumonisins and ochratoxin A) are covered by EU legislation (regulation or recommendation). Transgressions of these limits are rarely observed in official monitoring programs. However, low level contamination by Fusarium toxins is very common (e.g., deoxynivalenol (DON) is typically found in more than 50% of the samples) and co-contamination is frequently observed. Multi-mycotoxin studies reported 75%–100% of the samples to contain more than one mycotoxin which could impact animal health at already low doses. Co-occurrence of mycotoxins is likely to arise for at least three different reasons (i) most fungi are able to simultaneously produce a number of mycotoxins, (ii) commodities can be contaminated by several fungi, and (iii) completed feed is made from various commodities. In the present paper, we reviewed the data published since 2004 concerning the contamination of animal feed with single or combinations of mycotoxins and highlighted the occurrence of these co-contaminations.     

Deoxynivalenol (Vomitoxin)-Induced Anorexia Is Induced by the Release of Intestinal Hormones in Mice

Deoxynivalenol (DON), also known as vomitoxin, is a mycotoxin that can cause antifeeding and vomiting in animals. However, the mechanism of DON inducing anorexia is complicated. Studies have shown that intestinal hormones play a significant part in the anorexia caused by DON. We adopted the “modeling of acute antifeeding in mice” as the basic experimental model, and used two methods of gavage and intraperitoneal injection to explore the effect of intestinal hormones on the antifeedant response induced by DON in mice. We found that 1 and 2.5 mg/kg·bw of DON can acutely induce anorexia and increase the plasma intestinal hormones CCK, PYY, GIP, and GLP-1 in mice within 3 h. Direct injection of exogenous intestinal hormones CCK, PYY, GIP, and GLP-1 can trigger anorexia behavior in mice. Furthermore, the PYY receptor antagonist JNJ-31020028, GLP-1 receptor antagonist Exendin(9-39), CCK receptor antagonist Proglumide, GIP receptor antagonist GIP(3-30)NH₂ attenuated both intestinal hormone and DON-induced anorectic responses. These results indicate that intestinal hormones play a critical role in the anorexia response induced by DON.     

Efficient Adsorption of Deoxynivalenol by Porous Carbon Prepared from Soybean Dreg

A novel porous carbon adsorbent for the removal of deoxynivalenol was prepared from soybean dreg (SD). The new material was characterized by scanning electron microscopy equipped with energy dispersive X-ray spectroscopy (SEM-EDS), transmission electron microscopy (TEM), Brunauer–Emmett–Teller (BET) analysis, N₂ adsorption/desorption measurement techniques, X-ray diffraction (XRD), Raman spectroscopy, Fourier transform infrared (FTIR) spectroscopy and X-ray photoelectron spectroscopy (XPS). The specific surface area of the SDB-6-KOH was found to be 3655.95 m² g⁻¹, the pore volume was 1.936 cm³ g⁻¹ and the average pore size was 2.125 nm. The high specific surface area and effective functional groups of the carbon material promoted the adsorption of deoxynivalenol. By comparing the adsorption effect of SDB-6-X prepared with different activators (X: KOH, K₂CO₃, KHCO₃), SDB-6-KOH had the highest adsorption capacity. The maximum adsorption capacity of SDB-6-KOH to deoxynivalenol was 52.9877 µg mg⁻¹, and the removal efficiency reached 88.31% at 318 K. The adsorption kinetic and isotherm data were suitable for pseudo-second-order and Langmuir equations, and the results of this study show that the novel carbon material has excellent adsorptive ability and, thus, offers effective practical application potential for the removal of deoxynivalenol.     

Aflatoxins in Feed: Types,Metabolism, Health Consequences in Swine and Mitigation Strategies

Feeding farm animals with aflatoxin-contaminated feed can cause various severe toxic effects, leading to increased susceptibility to infectious diseases and increased mortality, weight loss, poor performance and reduced reproductive capability. Following ingestion of contaminated foodstuffs, aflatoxins are metabolized and biotransformed differently in animals. Swine metabolism is not effective in detoxifying and excreting aflatoxins, meaning the risk of aflatoxicosis is increased. Thus, it is of great importance to elucidate the metabolism and all metabolic pathways associated with this mycotoxin. The damage induced by AFB1 in cells and tissues consists of inhibition of cell proliferation, carcinogenicity, immunosuppression, mutagenicity, oxidative stress, lipid peroxidation and DNA damage, leading to pathological lesions in the liver, spleen, lymph node, kidney, uterus, heart, and lungs of swine. At present, it is a challenging task and of serious concern to completely remove aflatoxins and their metabolites from feedstuff; thus, the aim of this study was a literature review on the deleterious effects of aflatoxins on swine metabolism, as well as alternatives that contribute to the detoxification or amelioration of aflatoxin-induced effects in farm animal feed.     

Reduction of Mycotoxigenic Fungi Growth and Their Mycotoxin Production by Bacillus subtilis QST 713

The use of chemical pesticides to control the occurrence of mycotoxigenic fungi in crops has led to environmental and human health issues, driving the agriculture sector to a more sustainable system. Biocontrol agents such as Bacillus strains and their antimicrobial metabolites have been proposed as alternatives to chemical pesticides. In the present work, a broth obtained from a commercial product containing Bacillus subtilis QST 713 was tested for its ability to inhibit the growth of mycotoxigenic fungi as well as reduce their mycotoxin production. Mass spectrometry analysis of Bacillus subtilis broth allowed to detect the presence of 14 different lipopeptides, belonging to the iturin, fengycin, and surfactin families, already known for their antifungal properties. Bacillus subtilis broth demonstrated to be a useful tool to inhibit the growth of some of the most important mycotoxigenic fungi such as Aspergillus flavus, Fusarium verticillioides, Fusarium graminearum, Aspergillus carbonarius, and Alternaria alternata. In addition, cell-free Bacillus subtilis broth provided the most promising results against the growth of Fusarium graminearum and Alternaria alternata, where the radial growth was reduced up to 86% with respect to the untreated test. With regard to the mycotoxin reduction, raw Bacillus subtilis broth completely inhibited the production of aflatoxin B1, deoxynivalenol, zearalenone, and tenuazonic acid. Cell-free broth provided promising inhibitory properties toward all of the target mycotoxins, even if the results were less promising than the corresponding raw broth. In conclusion, this work showed that a commercial Bacillus subtilis, characterized by the presence of different lipopeptides, was able to reduce the growth of the main mycotoxigenic fungi and inhibit the production of related mycotoxins.     

In Vitro Metabolism of Phenylspirodrimanes Derived from the Indoor Fungus Stachybotrys

Fungi belonging to the genus Stachybotrys are frequently detected in water-damaged indoor environments, and a potential correlation between emerging health problems of inhabitants of affected housing and the fungi is controversially discussed. Secondary metabolites (i.e., mycotoxins) produced by Stachybotrys, such as the highly toxic macrocyclic trichothecenes (MCTs), are of potential concern to human health. The present study, however, focused on the potential effects of the more broadly and abundantly formed group of phenylspirodrimanes (PSDs). The phase I and II metabolism of four structurally different PSDs were investigated in vitro using hepatic models in combination with high-performance liquid chromatography high-resolution mass spectrometry (HPLC-HRMS) analysis. In addition to metabolite detection by HRMS, isolation and structure elucidation by nuclear magnetic resonance spectroscopy (NMR) was part of the conducted study as well.     

A Preliminary Study to Classify Corn Silage for High or Low Mycotoxin Contamination by Using near Infrared Spectroscopy

Mycotoxins should be monitored in order to properly evaluate corn silage safety quality. In the present study, corn silage samples (n = 115) were collected in a survey, characterized for concentrations of mycotoxins, and scanned by a NIR spectrometer. Random Forest classification models for NIR calibration were developed by applying different cut-offs to classify samples for concentration (i.e., μg/kg dry matter) or count (i.e., n) of (i) total detectable mycotoxins; (ii) regulated and emerging Fusarium toxins; (iii) emerging Fusarium toxins; (iv) Fumonisins and their metabolites; and (v) Penicillium toxins. An over- and under-sampling re-balancing technique was applied and performed 100 times. The best predictive model for total sum and count (i.e., accuracy mean ± standard deviation) was obtained by applying cut-offs of 10,000 µg/kg DM (i.e., 96.0 ± 2.7%) or 34 (i.e., 97.1 ± 1.8%), respectively. Regulated and emerging Fusarium mycotoxins achieved accuracies slightly less than 90%. For the Penicillium mycotoxin contamination category, an accuracy of 95.1 ± 2.8% was obtained by using a cut-off limit of 350 µg/kg DM as a total sum or 98.6 ± 1.3% for a cut-off limit of five as mycotoxin count. In conclusion, this work was a preliminary study to discriminate corn silage for high or low mycotoxin contamination by using NIR spectroscopy.     

Detoxification of the Mycotoxin Citrinin by a Manganese Peroxidase from Moniliophthora roreri

Citrinin (CIT) is a mycotoxin found in foods and feeds and most commonly discovered in red yeast rice, a food additive made from ordinary rice by fermentation with Monascus. Currently, no enzyme is known to be able to degrade CIT effectively. In this study, it was discovered that manganese peroxidase (MrMnP) from Moniliophthora roreri could degrade CIT. The degradation appeared to be fulfilled by a combination of direct and indirect actions of the MrMnP with the CIT. Pure CIT, at a final concentration of 10 mg/L, was completely degraded by MrMnP within 72 h. One degradation product was identified to be dihydrocitrinone. The toxicity of the CIT-degradation product decreased, as monitored by the increased survival rate of the Caco-2 cells incubated with MrMnP-treated CIT. In addition, MrMnP could degrade CIT (with a starting concentration of up to 4.6 mg/L) completely contaminated in red yeast rice. MrMnP serves as an excellent candidate enzyme for CIT detoxification.